Field of the Invention
The invention relates to methods of producing tobacco having reduced levels of tobacco specific nitrosamines in the cured leaves and transgenic tobacco plants for producing tobacco having reduced levels of tobacco specific nitrosamines in the cured leaves.
Description of the Related Art
In many organisms, antioxidant activity increases upon exposure to conditions that increase the formation of active oxygen species such as hydrogen peroxide. There is evidence that active oxygen species are ubiquitous messengers in plants that stimulate defense responses against stressful conditions.
It has been reported that tobacco specific nitrosamines (TSNAs) are produced primarily in the curing process. It is believed that antioxidants interfere with the nitrosation of secondary alkaloids thereby reducing the formation of TSNAs. Commonly owned U.S. application Ser. No. 10/235,636 discloses a method for reducing tobacco specific nitrosamine content in cured tobacco by increasing the levels of antioxidants in the tobacco prior to harvesting. The methods disclosed in that application include root pruning prior to harvesting, severing the xylem tissue prior to harvesting, and administering antioxidants or chemicals that produce an increase in antioxidants to the tobacco plant after harvesting. U.S. application Ser. No. 10/235,636 and all publications referred to below are hereby incorporated into the present application entirely and for all purposes.
U.S. Pat. No. 5,516,671 discloses that plants, for example potatoes, can be transformed with a gene encoding glucose oxidase to produce plants that are resistant to disease. See also, Wu et al. (Plant Physiology, 115:427-435, 1997).
Bema and Bernier (Plant Molecular Biology, 33:417-29, 1997) have shown that wheat germin has oxalate oxidase (OxO) activity and (Plant Molecular Biology, 39:539-49, 1999) that the promoter of the gemin gf-2.8 gene is active in transgenic tobacco. Indeed, most germins and many germin-like proteins have OxO activity. (Bemier and Bema, Plant Physiology and Biochemistry, 39:545-9, 2001). PCT Application Publication Number WO 99/04013 discloses the expression of oxidase enzymes as a method of producing resistance to stress in a variety of plants.
Published U.S. Application No. 2004/007218 discloses a method for determination of the level of H2O2 in a cell by measuring expression of a reporter polypeptide from a promoter that is inducible by H2O2.